The mammalian visual cortex is critical for vision and has been used as a model for the rest of the cerebral cortex. In spite of this, little is known about the detailed operations of its microcircuits. In fact, the cortex is composed of many different cell types, and, it is likely that each cell type has a particular circuit function. In the past cycle we focused on four major subtypes of cortical GABAergic interneurons (PV, SOM, VIP and chandeliers) in mouse visual cortex, using two- photon photoactivation and transgenic mice to map connections to and from interneurons in a systematic fashion. We found that interneurons often make synaptic connections with every single neighboring cell, and this promiscuity could be an important feature of the cortical inhibitory ?circuit blueprint?. In the course of these experiments we unexpectedly discovered that groups of coactive neurons, which we termed ?neuronal ensembles?, account for the majority of the cortical response to visual stimuli. Moreover, ensembles also dominate spontaneous activity and have distinct spatiotemporal characteristics. Further, using two-photon optogenetics we found by chance that ensembles can be artificially imprinted into the cortex and they can be recalled by stimulating individual neurons, showing pattern completion, even days after imprinting. These intriguing results suggest that ensembles could be multicellular building blocks of cortical function, implementing a population neural code. To pursue this discovery and test these ideas we now propose an experimental ?deep dive? into the properties of these ensembles, in order to characterize their basic phenomenology, understand their synaptic circuit mechanisms and test their role in the behavior of the animal in sensory discrimination. The project will be carried out in primary visual cortex of awake behaving mice and make use of a novel two-photon holographic microscopy method that enables us to image and optically manipulate neurons in different cortical layers simultaneously. Our work will provide a systematic anatomical and functional description of neuronal ensembles, how they regulate the activity of the cortex and how they can be manipulated and reconfigured. This could help in novel therapeutic strategies by reconfiguring pathological circuits and correct visual deficits.